Rat pancreas adenylate cyclase VII. Effect of extracellular calcium on pancreozymin-induced cyclic AMP formation

1. 1. The effect of stimulation of adenylate cyclase by pancreozymin-C-octapeptide on the cyclic AMP level of rat pancreatic fragments has been investigated.

2. 2. In normal Krebs-Ringer bicarbonate medium pancreozymin-C-octapeptide causes a slight increase in pancreatic cyclic AMP level; this increase can be considerably enhanced by incubation in a calcium-free incubation medium.

3. 3. The dose-responce curve for pancreazymin-C-octapeptide in calcium-free medium is shifted to lower peptide concentrations, compared to the curve in normal Krebs-Ringer bicarbonate medium.

4. 4. The maximal stimulatory effect of pancreozymin-C-octapeptide id obtained at a 1-methyl-3-isobutylxanthine concentration of 10 mM.

5. 5. It suffices to lower the Ca²⁺-concentration of the medium from 2.5 to 1.5 mM to get the maximal increase in cyclic AMP content under influence of pancreozymin-C-octapeptide.

6. 6. It is concluded that extracellular calcium antagonizes the stimulation of adenylate cyclase by pancreozymin-C-octapeptide. This suggest that a low cytoplasmic Ca²⁺-concentration is required for the maximal response of acinar cell adenylate cyclase to pancreozymin.

Absence of thrombin-activatable fibrinolysis inhibitor protects against sepsis-induced liver injury in mice

Thrombin-activatable fibrinolysis inhibitor (TAFI), also known as carboxypeptidase R, has been implicated as an important negative regulator of the fibrinolytic system. In addition, TAFI is able to inactivate inflammatory peptides such as complement factors C3a and C5a. To determine the role of TAFI in the hemostatic and innate immune response to abdominal sepsis, TAFI gene-deficient (TAFI-/-) and normal wild-type mice received an i.p. injection with Escherichia coli. Liver TAFI mRNA and TAFI protein concentrations increased during sepsis. In contrast to the presumptive role of TAFI as a natural inhibitor of fibrinolysis, TAFI-/- mice did not show any difference in E. coli-induced activation of coagulation or fibrinolysis, as measured by plasma levels of thrombin-anti-thrombin complexes and D-dimer and the extent of fibrin depositions in lung and liver tissues. However, TAFI-/- mice were protected from liver necrosis as indicated by histopathology and clinical chemistry. Furthermore, TAFI-/- mice displayed an altered immune response to sepsis, as indicated by an increased neutrophil recruitment to the peritoneal cavity and a transiently increased bacterial outgrowth together with higher plasma TNF-alpha and IL-6 levels. These data argue against an important part for TAFI in the regulation of the procoagulant-fibrinolytic balance in sepsis and reveals a thus far unknown role of TAFI in the occurrence of hepatic necrosis.     

Matrix metalloproteinase-9 deficiency impairs host defense against abdominal sepsis

Matrix metalloproteinase (MMP)-9 is involved in extracellular matrix degradation and leukocyte migration. To determine the role of MMP-9 in the innate immune response to peritonitis, MMP-9 gene-deficient (MMP-9(-/-)) and normal wild-type mice were i.p. infected with Escherichia coli. MMP-9 mRNA and pro-MMP-9 protein levels increased rapidly upon induction of peritonitis. Although MMP-9(-/-) neutrophils showed a normal phagocytosis of E. coli in vitro, MMP-9(-/-) mice displayed a reduced resistance against E. coli peritonitis, as indicated by an enhanced bacterial outgrowth in the peritoneal cavity and increased dissemination of the infection. Furthermore, the cytokine response to LPS was not influenced by MMP-9 deficiency. However, during E. coli peritonitis, MMP-9(-/-) mice showed much higher peritoneal chemokine and cytokine levels compared with wild-type mice. Despite the increased local chemokine concentrations, MMP-9(-/-) mice displayed a diminished recruitment of leukocytes to the site of infection, indicating that cellular migration was impaired. Moreover, MMP-9(-/-) mice developed more severe distant organ damage during infection. These data suggest that MMP-9 is an essential component of an effective host response to E. coli peritonitis.     

Endogenous tissue-type plasminogen activator is protective during Escherichia coli-induced abdominal sepsis in mice

Sepsis is associated with enhanced production of tissue-type plasminogen activator (tPA). We investigated the function of endogenous tPA in the immune responses to Escherichia coli-induced abdominal sepsis using tPA gene-deficient (tPA(-/-)) and normal wild-type (WT) mice. tPA(-/-) mice demonstrated an impaired defense against E. coli peritonitis as indicated by higher bacterial loads at the primary site of the infection, enhanced dissemination, and reduced survival. The protective function of tPA was independent of plasmin since plasminogen gene-deficient (Plg(-/-)) mice were indistinguishable from WT mice. Relative to WT mice, tPA(-/-) mice demonstrated similar neutrophil counts in the peritoneal cavity despite much higher bacterial loads and higher local concentrations of neutrophil attracting chemokines, suggesting a reduced migratory response. In line, tPA(-/-) mice demonstrated a reduced thioglycolate-induced neutrophil influx into the peritoneal cavity and i.p. injection of WT mice with a replication-defective adenoviral vector expressing tPA caused an enhanced cell migration to the peritoneal cavity during E. coli peritonitis. These findings identify a novel protective function of tPA in abdominal sepsis caused by E. coli that seems independent of its role in the generation of plasmin.     

Genetic analysis of the capsid gene of genotype GII.2 noroviruses

Noroviruses (NoVs) are considered to be a major cause of acute nonbacterial gastroenteritis in humans. The NoV genus is genetically diverse, and genotype GII.4 has been most commonly identified worldwide in recent years. In this study we analyzed the complete capsid gene of NoV strains belonging to the less prevalent genotype GII.2. We compared a total of 36 complete capsid sequences of GII.2 sequences obtained from the GenBank (n = 5) and from outbreaks or sporadic cases that occurred in The Netherlands (n = 10) and in Osaka City, Japan (n = 21), between 1976 and 2005. Alignment of all capsid sequences did not show fixation of amino acid substitutions over time as an indication for genetic drift. In contrast, when strains previously recognized as recombinants were excluded from the alignment, genetic drift was observed. Substitutions were found at five informative sites (two in the P1 subdomain and three in the P2 subdomain), segregating strains into five genetic groups (1994 to 1997, 1999 to 2000, 2001 to 2003, 2004, and 2005). Only one amino acid position changed consistently between each group (position 345). Homology modeling of the GII.2 capsid protein showed that the five amino acids were located on the surface of the capsid and close to each other at the interface of two monomers. The data suggest that these changes were induced by selective pressure, driving virus evolution. Remarkably, this was observed only for nonrecombinant genomes, suggesting differences in behavior with recombinant strains.     

Genome-Scale Genotype-Phenotype Matching of Two Lactococcus lactis Isolates from Plants Identifies Mechanisms of Adaptation to the Plant Niche

Lactococcus lactis is a primary constituent of many starter cultures used for the manufacturing of fermented dairy products, but the species also occurs in various nondairy niches such as (fermented) plant material. Three genome sequences of L. lactis dairy strains (IL-1403, SK11, and MG1363) are publicly available. An extensive molecular and phenotypic diversity analysis was now performed on two L. lactis plant isolates. Diagnostic sequencing of their genomes resulted in over 2.5 Mb of sequence for each strain. A high synteny was found with the genome of L. lactis IL-1403, which was used as a template for contig mapping and locating deletions and insertions in the plant L. lactis genomes. Numerous genes were identified that do not have homologs in the published genome sequences of dairy L. lactis strains. Adaptation to growth on substrates derived from plant cell walls is evident from the presence of gene sets for the degradation of complex plant polymers such as xylan, arabinan, glucans, and fructans but also for the uptake and conversion of typical plant cell wall degradation products such as α-galactosides, β-glucosides, arabinose, xylose, galacturonate, glucuronate, and gluconate. Further niche-specific differences are found in genes for defense (nisin biosynthesis), stress response (nonribosomal peptide synthesis and various transporters), and exopolysaccharide biosynthesis, as well as the expected differences in various mobile elements such as prophages, plasmids, restriction-modification systems, and insertion sequence elements. Many of these genes were identified for the first time in Lactococcus lactis. In most cases good correspondence was found with the phenotypic characteristics of these two strains.     

Complete resequencing and reannotation of the Lactobacillus plantarum WCFS1 genome

There is growing interest in the beneficial effects of Lactobacillus plantarum on human health. The genome of L. plantarum WCFS1, first sequenced in 2001, was resequenced using Solexa technology. We identified 116 nucleotide corrections and improved function prediction for nearly 1,200 proteins, with a focus on metabolic functions and cell surface-associated proteins.     

Evolution of prokaryotic subtilases: genome-wide analysis reveals novel subfamilies with different catalytic residues

Subtilisin-like serine proteases (subtilases) are a very diverse family of serine proteases with low sequence homology, often limited to regions surrounding the three catalytic residues. Starting with different Hidden Markov Models (HMM), based on sequence alignments around the catalytic residues of the S8 family (subtilisins) and S53 family (sedolisins), we iteratively searched all ORFs in the complete genomes of 313 eubacteria and archaea. In 164 genomes we identified a total of 567 ORFs with one or more of the conserved regions with a catalytic residue. The large majority of these contained all three regions around the "classical" catalytic residues of the S8 family (Asp-His-Ser), while 63 proteins were identified as S53 (sedolisin) family members (Glu-Asp-Ser). More than 30 proteins were found to belong to two novel subsets with other evolutionary variations in catalytic residues, and new HMMs were generated to search for them. In one subset the catalytic Asp is replaced by an equivalent Glu (i.e. Glu-His-Ser family). The other subset resembles sedolisins, but the conserved catalytic Asp is not located on the same helix as the nucleophile Glu, but rather on a beta-sheet strand in a topologically similar position, as suggested by homology modeling. The Prokaryotic Subtilase Database (www.cmbi.ru.nl/subtilases) provides access to all information on the identified subtilases, the conserved sequence regions, the proposed family subdivision, and the appropriate HMMs to search for them. Over 100 proteins were predicted to be subtilases for the first time by our improved searching methods, thereby improving genome annotation.     

Plasminogen activator inhibitor type-1-deficient mice have an enhanced IFN-gamma response to lipopolysaccharide and staphylococcal enterotoxin B

Plasminogen activator inhibitor type-1 (PAI-1) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Systemic inflammation is invariably associated with elevated circulating levels of PAI-1, and during human sepsis plasma PAI-1 concentrations predict an unfavorable outcome. Knowledge about the functional role of PAI-1 in a systemic inflammatory response syndrome is highly limited. In this study, we determined the role of endogenous PAI-1 in cytokine release induced by administration of LPS or staphylococcal enterotoxin B (SEB). Both LPS and SEB elicited secretion of PAI-1 into the circulation of normal wild-type (Wt) mice. Relative to Wt mice, PAI-1 gene-deficient (PAI-1(-/-)) mice demonstrated strongly elevated plasma IFN-gamma concentrations after injection of either LPS or SEB. In addition, PAI-1(-/-) splenocytes released more IFN-gamma after incubation with LPS or SEB than Wt splenocytes. Both PAI-1(-/-) CD4+ and CD8+ T cells produced more IFN-gamma upon stimulation with SEB. LPS-induced IFN-gamma release in mice deficient for uPA, the uPA receptor, or tPA was not different from IFN-gamma release in LPS-treated Wt mice. These results identify a novel function of PAI-1 during systemic inflammation, where endogenous PAI-1 serves to inhibit IFN-gamma release by a mechanism that does not depend on its interaction with uPA/uPA receptor or tPA.